Solid materials with charged surfaces are used widely in the field of protein purification generally, and for antibody purification in particular. These materials commonly include so-called ion exchangers, which are usually employed in either of two application formats. In bind-elute mode, the sample and ion exchanger are equilibrated to conditions that allow the antibody to bind. Contaminants that interact weakly or not at all with the charged surface fail to bind and are eliminated. Contaminants that interact more strongly than the antibody bind more strongly. After washing to remove unbound contaminants, the column may be eluted by increasing the salt concentration. This permits fractionation of bound species in increasing order of the strength of their interaction with the ion exchanger, thereby achieving a high degree of antibody purification. Inflow-through mode, the sample and ion exchanger are both equilibrated to conditions that prevent the antibody from binding. Species that interact more strongly with the ion exchanger than the antibody are bound and thereby removed, but species that bind more weakly than the antibody flow through with it and persist as contaminants. With human, humanized or chimeric IgG monoclonal antibodies, most bind-elute applications are conducted on cation exchangers (negatively charged surface). Most flow-through applications are conducted on anion exchangers (positively charged surface). Both modes are performed on charged surfaces presented in a variety of solid phase architectures, including porous or non-porous particles packed in columns, or added directly to large volume aqueous samples, or on monoliths or membranes. These different architectures confer different flow properties, capacity, and resolution, but the defining chemical features of flow-through or bind-elute chromatography are constant regardless of physical format. Both methods rely on the equilibration of the ion exchanger and sample to the same conditions before the sample is introduced to the column.
A method called High Performance Tangential Flow Filtration (HPTFF) has been described in which a positive charge is created on the surface of an ultrafiltration membrane with a size cutoff of about 100 to about 300 kDa (van Reis et al., J. Membr. Sci. (2007) 297:16-et seq.; van Reis U.S. Pat. No. 7,001,550; van Reis et al., J. Membr. Sci. (1999) 159:133-et seq.; Bolton et al., Adv. Filtr. Sep. Technol. (1999) 13A:537-et seq.; Burns et al., J. Membr. Sci. (1999) 64:27-et seq.; Mehta et al., CEP (2008) 104(5):514-et seq.). When a sample of crude IgG monoclonal antibody is introduced within a narrow range of pH and conductivity, IgG is repelled from the membrane surface and thus prevented from passing through the pores. The majority of contaminant species either binds to the surface or passes through the pores by convective mass transport, and is thereby eliminated. The method also permits concentration of the antibody, although such concentration relies on antibody equilibration to the operating conditions before it is applied to the membrane. Those operating conditions generally consist of weakly alkaline to near-neutral pH and low conductivity. Excessive conductivity may block electrostatic interactions and cause IgG loss through the membrane pores. The capacity of the method may be restricted by the proportion of acidic contaminants that bind to the membrane, since they neutralize the charge on the membrane. This may weaken or suspend antibody repulsion and cause antibody to be lost by passing through the pores with contaminants. Thus, like bind-elute and flow-through applications on traditional ion exchangers, HPTFF relies on equilibration of the sample and operating conditions in advance of performing the technique. Currently, HPTFF is employed solely in membrane applications. Its fluid-recycling approach may preclude its application to monoliths or columns of packed particles.
Another method employs mixed mode chromatography media with combinations of chemical functionalities (Eriksson et al., Bioprocess. Intl. (2009) 7:52-et seq.; Bresolin et al., J. Chromatogr. B (2010) 878:2087-et seq.; de Souza et al., J. Chromatogr. B (2010) 878:557-et seq.). Some of these media materials include positive charges and are applied in the same format as ion exchangers, i.e. in bind-elute and flow-through mode, although under different chemical conditions depending on the nature of the secondary functionalities. Yet another method is to combine physical functionalities with chemical functionalities. One such example employs variable size exclusion functionality along with porous particle anion exchange materials (Hunter et al., J. Chromatogr. A (2000) 897:65-et seq.; Hunter et al., J. Chromatogr. A (2000) 897:87-et seq.; Hunter et al., J. Chromatogr. A (2001) 930:79-et seq.; Hunter et al., J. Chromatogr. A (2002) 971:105-et seq.). The method generally involves entry of proteins into particle pores in a size-dependent manner while also exhibiting dependence on the charge on the protein, as well as the buffer conditions. The method has been used to bind and elute an IgG-type antibody, although it has not been employed in purification or to reduce antibody aggregates.
Positively charged soluble polymers (polyallylamine, polyarginine) and certain divalent cations (ethacridine, metal ions) have been employed to co-precipitate negatively charged contaminants from antibody preparations (Thömmes et al., in: U. Gottschalk (ed.), Process Scale Purification of Antibodies, J. Wiley and Sons, Hoboken, (2009) 293-et seq.; Ma et al., J. Chromatogr. B (2010) 878:798-et seq.; Peram et al., Biotechnol. Progr., (2010) 26:1322-et seq.; Glynn, in U. Gottschalk (ed.), Process Scale Purification of Antibodies, J. T. Wiley and Sons, Hoboken, (2009) 309-et seq.; Farhner et al., U.S. Patent Application No. 20080193981; Ma et al., J. Chromatogr. B (2010) 878:798-et seq.; Cordes et al., Biotechnol. Progr., (1990) 6:283-et seq.; Dissing, et al., Bioseparation, (1999), 7:221-et seq.; Bernhardt U.S. Pat. No. 5,559,250; Akcasu et al., Nature, (1960) 187:323-et seq.; Matsuzawa, et al., Nucl. Acids Res., (2003) 3(3):163-et seq.; Christensen et al., Prot. Expr. Purif., (2004) 37:468-et seq.; Kejnovsky et al., Nucl. Acids Res., (1997) 25:1870-et seq.; Ongkudon et al., Anal. Chem., (2011) 83:391-et seq.). These methods can be thought of as liquid-phase analogues to positively charged particles. Such methods can be carried out in an alternate physical format, commonly referred to as batch mode, in which the polymers are added directly to an antibody preparation within a narrow range of carefully controlled pH and conductivity conditions. Such variant methods have been employed in the selective precipitation of acidic host proteins from cell culture supernatants, as well as DNA, endotoxin, and virus.
Another issue that has been indicated is that unnatural hetero-aggregates can form spontaneously between host cell-derived contaminants and recombinant proteins produced by in vitro cell culture methods (Shukla et al., Biotechnol. Progr. (2008) 24:1115-et seq.; Luhrs, et al., J. Chromatogr. B (2009) 877:1543-et seq.; Mechetner et al., J. Chromatogr. B (2011) 879:2583-et seq.; Gagnon et al., J. Chromatogr. A, (2011) 1218:2405-et seq.; Gagnon, Bioprocessing J. (2010) 9(4):14-et seq.). These hetero-aggregates may be considered unnatural in two respects: 1) constituent contaminants are often of non-human origin, secreted by living non-human host cells or released into the culture media when non-human host cells lyse upon death. In living humans, such non-human contaminants do not exist; and 2) constituent contaminants accumulate to high concentrations in comparison to human in vivo systems where dead cell constituents are quickly eliminated. Accordingly, recombinant products are exposed to high levels of strongly interactive contaminants at concentrations that typically do not occur in living systems. Meanwhile, high expression levels of recombinant proteins make them suitable substrates for non-specific associations with these non-human contaminants, favoring the formation of undesirable hetero-aggregates of diverse composition.
The contaminating protein content of hetero-aggregates has been addressed to some extent via direct targeting of the contaminating protein (Shukla et al. and Gagnon et al. supra), as well as indirectly via targeting of the corresponding DNA component responsible for the contaminating protein (Luhrs et al. and Gagnon supra). A reduction of antibody aggregate level has been indicated when some complexes are dissociated (Shukla et al., Mechetner et al., and Gagnon supra). The ability of anion exchangers to reduce levels of antibody-contaminant complexes has been disclosed (Luhrs et al. and Gagnon et al. supra), but no study has revealed an anion exchange treatment that was able to fully eliminate hetero-aggregates. Size exclusion, cation exchange, and hydrophobic interaction chromatography were all generally inferior to anion exchange (Gagnon et al. supra).
Treating antibody preparations with agents that might be expected to dissociate hetero-aggregates has generally proven ineffective. For example, employing high concentrations of urea, salts, or combinations of the two does not substantially dissociate IgM-contaminant hetero-aggregates (Gagnon et al. supra). Protein A affinity chromatography with pre-elution washes of urea, alcohol, and surfactants has been indicated to reduce hetero-aggregate levels more effectively than without washes (Shukla et al. supra), as did pre-elution washes combining urea, salt, and EDTA with protein G affinity chromatography (Mechetner et al. supra). Anion exchange chromatography with a pre-elution wash of urea has been indicated to reduce hetero-aggregates more effectively than in the absence of a urea wash (Mechetner et al. supra). Cation exchange chromatography has also been indicated to reduce hetero-aggregates more effectively with a pre-elution EDTA wash than without the wash (Mechetner et al. supra). Finally, hydroxyapatite with pre-elution washes of urea and/or salt have also reduced hetero-aggregates more effectively than without such washes (Gagnon supra). Despite these observations, in general, the use of dissociating agents in pre-elution washes of antibodies bound to chromatography columns has been only moderately successful.